Developing a microfluidic-based epicPCR reveals diverse potential hosts of the mcrA gene in marine cold seep

Anaerobic methanotrophic (ANME) microbes play a crucial role in the bioprocess of anaerobic oxidation of methane (AOM). However, due to their unculturable status, their diversity is poorly understood. In this study, we established a microfluidics-based epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) to fuse the 16S rRNA gene and mcrA gene to reveal the diversity of ANME microbes (mcrA gene hosts) in three sampling push-cores from the marine cold seep. A total of 3725 16S amplicon sequence variants (ASVs) of the mcrA gene hosts were detected, and classified into 78 genera across 23 phyla. Across all samples, the dominant phyla with high relative abundance (>10%) were the well-known Euryarchaeota, and some bacterial phyla such as Campylobacterota, Proteobacteria, and Chloroflexi; however, the specificity of these associations was not verified. In addition, the compositions of the mcrA gene hosts were significantly different in different layers, where the archaeal hosts increased with the depths of sediments, indicating the carriers of AOM were divergent in depth. Furthermore, the consensus phylogenetic trees of the mcrA gene and the 16S rRNA gene showed congruence in archaea not in bacteria, suggesting the horizontal transfer of the mcrA gene may occur among host members. Finally, some bacterial metagenomes were found to contain the mcrA gene as well as other genes that encode enzymes in the AOM pathway, which prospectively propose the existence of ANME bacteria. This study describes improvements for a potential method for studying the diversity of uncultured functional microbes and broadens our understanding of the diversity of ANMEs.